Our objective is to define the initial, intracellular events of glucocorticoid hormone and steroid hormone action in general. Such studies are relevant since steroid receptors are arguably the best understood regulators of eukaryotic gene transcription. A major unresolved issue in steroid hormone action is what controls the activity of a given ligand bound to its cognate, intracellular receptor. One hypothesis is that ligand-induced conformational changes in the C-terminus of receptors, as revealed by partial proteolysis, might determine the agonist vs antagonist activity of the receptor-steroid complex. We have found that steroid binding does cause a conformational change in glucocorticoid receptors (GRs), as detected by partial trypsin digestion. However, no uniformity of digestion patterns was found for unactivated or activated receptors bound with a series of six structurally different antagonists. Using a series of point mutations and epitope-specific antibodies, it was determined that the various tryptic fragments arose from C-terminal and/or internal cleavages. Thus, the presence or absence of C-terminal amino acids of the GR did not uniquely determine either the appearance of smaller trypsin-resistant fragments or the nature of the biological response of receptor-bound antisteroids. An unusual parameter that has been described to influence the activity of GR complexes is a 21 bp sequence, located at -3.6 kb of the rat tyrosine aminotransferase (TAT) gene, which causes both a left shift in the dose-response curve of agonists and an increase the partial agonist activity of antagonists. Because this behaviour is seen both with the endogenous TAT gene and synthetic reporter genes, the 21 bp element has been called a glucocorticoid modulatory element, or GME. We have now characterized the neighboring TAT gene sequences and find that two other elements cooperate with the GME to afford the full activity of the native gene: a negative element at -3.1 kb and a neutralizing sequence between -2.6 and -2.3 kb. Such a grouping of elements appears to be novel among steroid regulated genes but is a not uncommon in other systems. The activities of this complex grouping can be reproduced by constructs containing just the GME and synthetic promoter and reporters, thereby simplifying future studies with the two GME binding proteins that we have recently cloned. Collectively, our findings contribute to our long term goal of defining the action of steroid hormones at a molecular level and of understanding their role in human physiology.